We will continue our investigation of the structure and replication of yeast DNA molecules. We have shown previously that each yeast chromosome is composed of a single DNA duplex. For the smallest chromosomes the mass of the duplex is about 10 to the 8th power daltons, and for the largest about 10 to the 9th power daltons. An attempt will be made to identify DNA from the smallest chromosome with a specific linkage group by comparing sedimentation profiles of disomic and normal euploid strains. Previous work has shown that, as with animal chromosomes, each yeast chromosome contains a number of DNA replication initiation sites. We will try to construct a replication map of the smallest yeast chromosome with a technique similar to that used for bacteriophage lambda. This will involve comparing the distribution of replication structures on replication intermediates of chromosomes of the smallest size class. The relationship of mitochondrial and nuclear DNA replication will be examined by determining whether mitochondrial DNA replication is affected by a variety of mutations which block nuclear DNA replication. The results should give some insight into the extent of metabolic and regulatory coupling between these two systems of replication. We will also attempt to determine the distribution of replication events among the 20 mitochondrial DNA molecules in a cell, i.e., whether all or only a fraction of the molecules engage in replication during a single cell cycle.